Laboratory of Biophysical Chemistry, Kyoto Pharmaceutical University, 5 Misasagi-Nakauchi-cho, Yamashina-ku, Kyoto, 607-8414, Japan.
Department of Bioanalytical Chemistry, Kobe Pharmaceutical University, 4-19-1 Motoyama-Kitamachi, Higashinada-ku, Kobe, 658-8558, Japan.
Sci Rep. 2023 Jul 5;13(1):10885. doi: 10.1038/s41598-023-38070-4.
Regulation of α-synuclein (αS) fibril formation is a potent therapeutic strategy for αS-related neurodegenerative disorders. αS, an intrinsically disordered 140-residue intraneural protein, comprises positively charged N-terminal, hydrophobic non-amyloid β component (NAC), and negatively charged C-terminal regions. Although mouse and human αS share 95% sequence identity, mouse αS forms amyloid fibrils faster than human αS. To evaluate the kinetic regulation of αS fibrillation, we examined the effects of mismatched residues in human and mouse αS on fibril formation and intramolecular interactions. Thioflavin T fluorescence assay using domain-swapped or C-terminal-truncated αS variants revealed that mouse αS exhibited higher nucleation and fibril elongation than human αS. In mouse αS, S87N substitution in the NAC region rather than A53T substitution is dominant for enhanced fibril formation. Fӧrester resonance energy transfer analysis demonstrated that the intramolecular interaction of the C-terminal region with the N-terminal and NAC regions observed in human αS is perturbed in mouse αS. In mouse αS, S87N substitution is responsible for the perturbed interaction. These results indicate that the interaction of the C-terminal region with the N-terminal and NAC regions suppresses αS fibril formation and that the human-to-mouse S87N substitution in the NAC region accelerates αS fibril formation by perturbing intramolecular interaction.
α-突触核蛋白（αS）纤维形成的调节是治疗与 αS 相关的神经退行性疾病的有效策略。αS 是一种内源性无序的 140 个残基的神经内蛋白，由带正电荷的 N 端、疏水性非淀粉β成分（NAC）和带负电荷的 C 端组成。尽管小鼠和人类 αS 有 95%的序列同一性，但小鼠 αS 形成淀粉样纤维的速度比人类 αS 快。为了评估 αS 纤维化的动力学调节，我们研究了人源和鼠源 αS 中的错配残基对纤维形成和分子内相互作用的影响。使用域交换或 C 端截断的 αS 变体的硫黄素 T 荧光测定法表明，与人类 αS 相比，小鼠 αS 具有更高的成核和纤维延伸能力。在小鼠 αS 中，NAC 区域中的 S87N 取代而不是 A53T 取代是增强纤维形成的主要原因。Förster 共振能量转移分析表明，在人类 αS 中观察到的 C 端与 N 端和 NAC 区域的分子内相互作用在小鼠 αS 中受到干扰。在小鼠 αS 中，S87N 取代是导致相互作用紊乱的原因。这些结果表明，C 端区域与 N 端和 NAC 区域的相互作用抑制了 αS 纤维形成，而 NAC 区域的人源到鼠源 S87N 取代通过干扰分子内相互作用加速了 αS 纤维形成。
